| We previously identified LncRNA NCk1-AS1 as a diagnostic marker for cervical carcinoma that located at chromosome 3q22.3.The NCK1-AS1 gene was found to be transcribed according to ENCODE chromosome modification data and ChromHMM chromosome state segmentation data.We founded that NCK1-AS1 was over-expressed in cervical cancer tissue compared with nornal tissue,indicate NCK1-AS1 can promote the development of tumor,and however,the underlying mechanism remained elusive.In the present study,we investigate the expression of NCK1-AS1,the function and its molec?lar mechanism in colon cancer.Part 1: The expression of NCK1-AS1 and clinical significance analysis in cervical cancerObjectives: To detect the expression of NCK1-AS1 in cervical cancer tissues and normal tissues.The relationship between NCK1-AS1 expression level and clinicopathologic features was analyzed.Methods: To detection of prognostic lncRNAs that are involved in cervical cancer progression,we subjected analysis of TCGA Cervical squamous cell carcinoma and endocervical adenocarcinoma RNA sequencing data by univariable Cox proportional hazards regression analysis.To validate the expression of NCK1-AS1 in cervical cancer,qRT-PCR and RNA in situ hybridization(RISH)assay were performed to detect the level of NCK1-AS1 in 31 paired CC tissues and adjacent cancer normal tissues.The statistical analyses were used for relationship with the pathologic grade,clinical stage and size of the tumour,lymph node metastasis.Res?lts: We identified a set of four-lncRNA signature that was significantly correlated with patients' survival.Res?lts confirmed that NCK1-AS1 was highly up-reg?lated(P< 0.01)in 77.4%(24/31)in CC tissues compared with normal tissues.Statistical analysis showed no correlations between NCK1-AS1 expression and age,lymph node numbers,tumor size,or clinical stage(P>0.05).Interestingly,NCK1-AS1 expression level was significantly associated with histological type(P<0.05)and lymph node status(P<0.05)Conclusion: LncRNA NCK1-AS1 is specifically up-reg?lated in cervical cancer and associate with clinical progression.Part 2: The function of NCK1-AS1 in the cell cycle and progression in cervical cancerObjectives: To study the effects of NCK1-AS1 on cell proliferation,cell cycle,invasion,colony formation incervical cancer cell CaSki?SiHa and HeLa,and tumorigenesis in vivo.Methods: To identify the core promoter region of NCK1-AS1 gene,four variety luciferase reporter constructs containing the overlapped different fragments of NCK1-AS1 gene 1000 bp region upstream were constructed,as flowing D1000(-1000-0),D750(-750-0),D500(-500-0)and D250(-250-0).These four luciferase reporter constructs were transfected SiHa cells and after 48 h cells were lysed and their luciferase activities were measured.To validate the function of NCK1-AS1 in reg?lating cervical cancer cell phenotype,knockdown of NCK1-AS1 in CaSki and SiHa cells that with higher NCK1-AS1 expression were carried out via siRNA/shRNA mediated silencing and cell cycle profile and proliferation were subsequently analyzed.qRT-PCR analysis confirmed that the NCK1-AS1 expression levels was significantly knockdown in two cell lines.To further validate the role of NCK1-AS1 in the tumorigenesis of cervical cancer,NCK1-AS1 stable knockdown CaSki cells or control cells were injected into nude mice.Res?lts: Dual-Luciferase assay showed that significant increase in luciferase activities was detectable in cells transfected with D1000,D750,D500 and D250 compared with the pGL3-basic group.These data demonstrated that a genomic region from-750 to-250 of NCK1-AS1 gene has a strong promoter activity.FCM and EdU demonstrated that NCK1-AS1 affected the G1-S transition of cell-cycle progression and inhibited the proliferation,migration and invasion of CC cells.At the end of this experiment,tumor weight of NCK1-AS1 stable knockdown group(0.683±0.121g)was only 7% of the control group(0.053±0.015 g),Conclusion: Interestingly,we demonstrated that transcription factor SP1 directly binding to the promoter to activation NCK1-AS1 expression in SiHa cells.In vitro and in vivo assays of silencing NCK1-AS1 significantly inhibited cell proliferation and invasion,which induces cell arrest in S phase of cell cycle.Part 3: the molec?lar mechanism of the cell cycle and progression of cervical cancer induced by NCK1-AS1Objectives: To verify the molec?lar mechanism of the development and progression of colon cancer induced by NCK1-AS1.Methods: To further explore the potential molec?lar mechanisms of NCK1-AS1 in CC cells,Human Transcriptome Array 2.0 analysis was performed to investigate the differential gene expression profiles between NCK1-AS1 knockdown group and control group in CaSki cells.For further confirmation,we constructed two dual-luciferase reporters containing:(1)Luc-NCK1-AS1-WT;(2)Luc-NCK1-AS1 MT(mutated on the putative miR-6857 sites).MiR-6857 over-expression slowed inhibits the propagation of the CC cells as analyzed using CCK8 assay.Transwell assays were performed to detect the role of miR-6857 on invasion and res?lts showed that overexpression miR-6857 decreased CaSki cell invasion,we constructed luciferase reporters containing the he putative miR-6857 binding sites,which contains wild-type(WT)or mutated miR-6857 binding sites.We found that overexpression of mi R-6857 reduced the luciferase activities of the WT reporter vector but not mutant reporter vector.Res?lts: Through bioinformatics' analysis,493 coding-genes and 413 non-coding genes were differentially expressed under the condition of “Q< 0.001 and fold change > 1.3”.As expected,overexpression of miR-6857 reduced the luciferase activities of the WT reporter vector but not empty vector or mutant reporter vector.Also,we detect whether the exogenous over-expression of NCK1-AS1 induces a more malignant phenotype to cervical cancer.Structures of f?ll-length and mutant(mutated on the putative miR-6857 sites)were generated.Hela cells constitutively GFP tagged f?ll-length NCK1-AS1(LV-NCK1-AS1),or GFP-tagged NCK1-AS1 lacking putative miR-6857 binding sites(LV-NCK1-AS1 MT).We next assessed the effects of NCK1-AS1 over-expression on invasion and cell proliferation.Res?lts showed that LV-NCK1-AS1 HeLa cells were significantly more invasive than both LV-NCK1-AS1 MT and wild type HeLa cells.Over-expression of f?ll-length NCK1-AS1 promoted the anchorage-independent growth of HeLa cells.Conclusion: In conclusion,these data indicate lncRNA NCK1-AS1 downreg?lated the RNA levels of miR-6857 through directly binding to them thereby derepresses CDK1 expression and imposes an additional level of post-transcriptional reg?lation. |
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